There is an ongoing need for compositions and methods that relate to prophylaxis and therapy of diseases that involve undesirable angiogenesis. Angiogenesis is in part related to expression of endoglin on endothelial cells.
Endoglin
Endoglin is a homodimeric tumor-associated cell membrane antigen with molecular size of 160 kilodaltons (kD) or 170 kD that is mainly expressed on leukemia cells and endothelial cells (Haruta and Seon, 1986, Proc. Natl. Acad. Sci. USA 83: 7898-7902; Gougos and Letarte, 1988, J. Immunol. 141: 1925-1933; Seon et al., 1997, Clin. Cancer Res. 3: 1031-1044). Endoglin's expression on endothelial cells is upregulated on proliferating endothelial cells of the tumor-associated vascular and lymphatic endothelium (Seon et al., Supra; Burrows et al., 1995, Clin. Cancer Res. 1: 1623-1634; Matsuno et al., 1999, Clin Cancer Res. 5: 371-382; Clasper et al., 2008, 68: 7293-7303). Endoglin is essential for angiogenesis/vascular development (Li et al., 1999, Science 284: 1534-1537; Arthur et al., 2000, Dev. Biol. 217: 42-53) and a co-receptor of TGF-β (Cheifetz et al., 1992, J. Biol. Chem. 267: 19027-19030).
Murine endoglin has been characterized as a dimer with molecular size of approximately 180 kilodaltons (kD). Human endoglin exists in two forms; i.e., a smaller 160 kD form and a larger 170 kD form with the difference between the two being found in the cytoplasmic portion of the protein. Endoglin has an extracellular region, a hydrophobic transmembrane region, and a cytoplasmic tail. A comparison of the nucleotide sequence of human endoglin with murine endoglin reveals an identity of about 71 to 72% (St. Jacques et al., 1994, Endocrinol. 134:2645-2657; Ge and Butcher, 1994, Gene 158:2645-2657). However, in the human and murine sequences encoding the transmembrane regions and cytoplasmic regions of endoglin, there is a 93-95% identity. Thus, in the human and murine sequences encoding the extracellular region to which antibody would be directed at the cell surface, there is significantly less identity than 70%. Although the amino acid sequence similarity between human and mouse endoglins appears substantial, the observed amino acid sequence differences in the extracellular regions should be sufficient for generating distinct antigenic epitopes unique to human endoglin or to mouse endoglin. This is because in peptide epitopes, even a subtle variation in the amino acid sequence comprising the epitopes or in the flanking amino acid sequences can markedly influence the immunogenicity of the epitopes (see, e.g., Vijayakrishnan et al., 1997, J. Immunol. 159:1809-1819). For instance, single amino acid substitutions in a peptide can cause marked changes in the immunogenicity of the peptide (Vijayakrishnan et al., 1997, supra). Such changes in a peptide epitope strongly influence the specificity of mAbs because mAbs define fine specificity.
Monoclonal Antibodies (mAbs) to Endoglin
SN6 is an antibody generated from immunization of mice with tumor-associated components of glycoprotein mixtures of cell membranes of human leukemia cells (Haruta and Seon, 1986, Proc. Natl. Acad. Sci. 83:7898-7902). It is a murine mAb that recognizes human endoglin. mAb 44G4 is an antibody generated from immunization of mice with whole cell suspensions of human pre-B leukemia cells (Gougos and Letarte, 1988, J. Immunol. 141:1925-1933; 1990, J. Biol. Chem. 265:8361-8364). It is a murine mAb that recognizes human endoglin. mAb MJ7/18 is an antibody generated from immunization of rats with inflamed mouse skins (Ge and Butcher, 1994, supra). It is a mAb that recognizes murine endoglin. mAb Tec-11 is an antibody generated from immunization of mice with human umbilical vein endothelial cells (Burrows et al., 1995, Clin. Cancer Res. 1:1623-1634). It is a murine mAb with reactivity restricted to human endoglin.
By the use of anti-endoglin antibodies and various staining procedures it has been determined that endoglin is expressed at moderate levels on human tumor cells such as from human leukemia, including non-T-cell-type (non-T) acute lymphoblastic leukemia (ALL), myelo-monocytic leukemia. In addition, it has been determined that endoglin is expressed at moderate to high levels in endothelial cells contained in tumor-associated vasculatures from human solid tumors, including angiosarcoma, breast carcinoma, cecum carcinoma, colon carcinoma, Hodgkins lymphoma, lymphoma, lung carcinoma, melanoma, osteosarcoma, ovarian carcinoma, parotid tumor, pharyngeal carcinoma, rectosigmoid carcinoma; and human vasculature from placenta. A lesser degree (weak) endothelial cell staining for endoglin has been observed in a variety of normal human adult tissue sections from spleen, thymus, kidney, lung and liver.
Increased endoglin expression on vascular endothelial cells has also been reported in pathological conditions involving angiogenesis. Such angiogenesis-associated diseases include most types of human solid tumors, rheumatoid arthritis, stomach ulcers, and chronic inflammatory skin lesions (e.g., psoriasis, dermatitis; Westphal et al., 1993, J. Invest. Dermatol. 100:27-34).
Angiogenesis
Angiogenesis includes the formation of new capillary blood vessels leading to neovascularization. Angiogenesis is a complex process which involves a series of sequential steps, including endothelial cell-mediated degradation of vascular basement membrane and interstitial matrices, migration of endothelial cells, proliferation of endothelial cells, and formation of capillary loops by endothelial cells. Solid tumors are angiogenesis-dependent; i.e., as a small solid tumor reaches a critical diameter, for further growth it needs to elicit an angiogenic response in the surrounding normal tissue. The resultant neovascularization of the tumor is associated with more rapid growth, and local invasion. Further, an increase in angiogenesis is associated with an increased risk of metastasis. Therefore, antiangiogenic therapy to inhibit tumor angiogenesis would suppress or arrest tumor growth and its spread.
In normal physiological processes such as wound healing, angiogenesis is turned off once the process is completed. In contrast, tumor angiogenesis is not self-limiting. Further, in certain pathological (and nonmalignant) processes, angiogenesis is abnormally prolonged. Such angiogenesis-associated diseases include diabetic retinopathy, chronic inflammatory diseases including rheumatoid arthritis, dermatitis, and psoriasis.
Antiangiogenic Therapy and Vascular Targeting Therapy of Human Solid Tumors
The progressive growth of solid tumors beyond clinically occult sizes (e.g., a few mm3) requires the continuous formation of new blood vessels, a process known as tumor angiogenesis. Tumor growth and metastasis are angiogenesis-dependent. A tumor must continuously stimulate the growth of new capillary blood vessels to deliver nutrients and oxygen for the tumor itself to grow. Therefore, either prevention of tumor angiogenesis (antiangiogenic therapy) or selective destruction of tumor's existing blood vessels (vascular targeting therapy) present a strategy directed to preventing or treating solid tumors.
Since a local network of new capillary blood vessels provide routes through which the primary tumor may metastasize to other parts of the body, antiangiogenic therapy should be important in preventing establishment of small solid tumors or in preventing metastasis (See, e.g., Folkman, 1995, Nature Medicine, 1:27-31). On the other hand, the vascular targeting therapy which attacks the existing vasculature is likely to be most effective on large tumors where the vasculature is already compromised (See, e.g., Bicknell and Harris, 1992, Semin. Cancer Biol. 3:399-407). Appropriately selected monoclonal antibodies can target vascular endothelial cells of tumors and can inhibit tumor growth and/or destroy tumors by several mechanisms that include antibody-dependent cell-mediated cytotoxicity (ADCC) and induction of apoptosis (Seon et al., 2011, Current Drug Delivery 8:135-143). In another case, monoclonal antibodies and fragments thereof can be used as a means of delivering to either existing tumor vasculature or newly forming tumor neovascularization therapeutic compounds in a method of antiangiogenic therapy and vascular targeting therapy (collectively referred to as “antiangiogenic therapy”). However, there remains a need for tools that can be used in identifying monoclonal antibodies that are likely to have effective clinical applications in humans, and there is accordingly a need for such antibodies themselves.
Mouse Models for Angiogenesis-Associated Diseases
The use of mouse models of angiogenesis has been accepted and validated for the evaluation of therapeutic agents because the models have been shown to reflect the clinical parameters characteristic of the respective disease, as well as predictive of the effectiveness of therapeutic agents in patients. These mouse models include, but are not limited to: mouse model for retinal neovascularization (Pierce et al., 1995, Proc. Natl. Acad. Sci. USA 92:905-909); mouse models for rheumatoid arthritis (MRL-lpr/lpr mouse model, Folliard et al., 1992, Agents Actions 36:127-135; mev mouse, Kovarik et al., 1994, J. Autoimmun 7:575-88); mouse models for angiogenesis (Majewski et al., 1994, Int. J. Cancer 57:81-85; Andrade et al., 1992, Int. J. EXp. Pathol., 73:503-13; Sunderkotter et al., 1991, Am. J. Pathol. 138:931-939); mouse model for dermatitis (Maguire et al., 1982, J. Invest. Dermatol. 79:147-152); and mouse model for psoriasis (Blandon et al., 1985, Arch. Dermatol. Res. 277:121-125; Nagano et al., 1990, Arch. Dermatol. Res. 282:459-462). Several anti-human endoglin (hENG) monoclonal antibodies (mAbs) are known to be potentially useful for antiangiogenic therapy. These mAbs include K4-2C10 (or termed SN6f), D4-2G10 (or termed SN6a), Y4-2F1 (or termed SN6j) and P3-2G8 (or termed SN6k). However, all of these mAbs cross-react with murine endoglin or murine endothelial cells. However, these cross-reactivities are weak and evaluation of these mAbs in animal (mice) for antiangiogenic activity can be done only under limited conditions (Seon et al., 2011). Therefore, there is a need for new animal models expressing human endoglin that can be targeted by more anti-human endoglin mAbs, as well as a need for new effective anti-human endoglin mAbs themselves. The present invention meets these and other needs.